1. Technical Field
The invention relates generally to optical methods and apparatus for measuring fluorescent substances in liquids and more particularly to methods and devices for measuring the fluorescence of chlorophyll, dyes and optical backscatter in natural water or in different solvents.
2. Description of the Prior Art
Chlorophyll a is the most widely used indicator of algal biomass for the purposes of long-term monitoring and management programs, as well as short-term research studies. Its degradation products (i.e., phaeophytin a and phaeophoribide a) can be used to assess the physiological health of algae and its fluorescence properties can be related directly to chlorophyll a concentration. More recently, the biochemical/biophysical characteristics of the chlorophyll-fluorescence process have led to techniques, which in addition to more accurately estimating algal biomass, can almost instantaneously estimate the rate of algal photosynthesis (i.e., growth).
Current laboratory analysis of chlorophyll a requires intensive processing. In-field or rapid laboratory filtration for pigment concentration and extraction, with organic solvents, is required. In recent years chromatographic procedures (HPLC) have been used to accurately resolve individual pigments. Nevertheless, these methods are expensive, difficult, and are limited in their availability from commercial laboratories, with this method used by a relatively few research groups. Furthermore, at present, there appears to be little potential for this technique to be used in-situ.
The most commonly utilized in-situ technologies for chlorophyll a determination are spectrophotometric and fluorometric methods of analysis. However, these methods contain absolute accuracy limitations due to relatively poor optical design, causing low spectral resolution for chlorophyll a. Problematic to these device development efforts is that absorption and emission maxims for chlorophylls b and c are close to the peak of chlorophyll a, in the red region of the spectrum, allowing for errors to occur when these pigments constitute a significant proportion of the total chlorophyll content and correction for these interferences are not taken into account. Additional errors may be introduced by various accessory pigments and degradation products as well as humic acid and fulvic acid color, which also absorb and fluoresce at wavelengths similar to those for chlorophyll a, b and c, causing significant error when analyzed using broadband florescent techniques. In addition to accessory pigment interference, when utilizing broadband fluorometric analysis of photosynthetic pigments, significant error may also occur from optical backscatter (i.e., suspended inorganic and non-fluorescent organic particles). All of these interferences need to be taken into account during calibration and in the optical and electronic design of fluorometric probes. The claims as part of this present innovation describe a novel optical design that maybe utilized to increase the accuracy and specificity of fluorometric probes.